Refinement ends up with a feature-less ball
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Hi everyone,
I converted the post-refinement results from relion and was trying to switch to frealign.
First I use the conversion script to convert relion star file to par file.(http://grigoriefflab.janelia.org/frealign_conversion_scripts)
Then I use relion_stack_create to generate the particle stack.
Frealign could run and finish without error (V9.11).
However, no matter how I changed the parameter, I could only get a feature-less ball.
I am not sure which step went wrong.
Any input will be greatly appreciated.
Below is my mparameters file.
Control parameter file to run Frealign
======================================
This file must me kept in the project working directory from which the refinement scripts are launched.
Note: Please make sure that project and scratch directories (if specified) are accessible by all sub-processes that are run on cluster nodes.
# Computer-specific setting
cluster_type none ! Set to "sge", "lsf", "slurm", "stampede", "pbs" or "condor" when running on a cluster, otherwise set to "none".
nprocessor_ref 64 ! Number of CPUs to use during refinement.
nprocessor_rec 8 ! Number of CPUs to use during reconstruction.
mem_per_cpu 8192 ! Memory available per CPU (in MB).
# Refinement-specific parameters
MODE 1 ! 1, 2, 3 or 4. Refinement mode, normally 1. Set to 2 for additional search.
start_process 2 ! First cycle to execute. Output files from previous cycle (n-1) required.
end_process 10 ! Last cycle to execute.
res_high_refinement 10.0 ! High-resolution limit for particle alignment.
res_high_class 8.0 ! High-resolution limit to calculate class membership (OCC).
nclasses 1 ! Number of classes to use.
DANG 200.0 ! Mode 3, 4 and 5: Angular step for orientational search.
ITMAX 200 ! Mode 2 and 4: Number of repetitions of grid search with random starting angles.
# Dataset-specific parameters
data_input Data ! Root name for parameter and map files.
raw_images stack.mrc
image_contrast P ! N or P. Set to N if particles are dark on bright background, otherwise set to P.
outer_radius 180.0 ! Outer radius of spherical particle mask in Angstrom.
inner_radius 0.0 ! Inner radius of spherical particle mask in Angstrom.
mol_mass 400.0 ! Molecular mass in kDa of particle or helical segment.
Symmetry C4 ! Symmetry of particle.
pix_size 2.6 ! Pixel size of particle in Angstrom.
dstep 10.0 ! Pixel size of detector in micrometer.
Aberration 2.7 ! Sherical aberration coefficient in millimeter.
Voltage 300.0 ! Beam accelleration voltage in kilovolt.
Amp_contrast 0.07 ! Amplitude contrast.
# Expert parameters (for expert users)
XSTD 0.0 ! Tighter masking of 3D map (XSTD > 0) or particles (XSTD < 0).
PBC 2.0 ! Discriminate particles with different scores during reconstruction. Small values (5 - 10) discriminate more than large values (50 - 100).
parameter_mask "1 1 1 1 1" ! Five flags of 0 or 1 (e.g. 1 1 1 1 1). Determines which parameters are refined (PSI, THETA, PHI, SHX, SHY).
refineangleinc 4 ! When larger than 1: Alternate between refinement of OCC and OCC + angles.
refineshiftinc 4 ! When larger than 1: Alternate between refinement of OCC and OCC + angles + shifts.
res_reconstruction 5.2 ! High-resolution limit of reconstruction. Normally set to Nyquist limit.
res_low_refinement 200.0 ! Low-resolution limit for particle alignment. Set to particle dimention or larger.
thresh_reconst 0.0 ! Particles with scores below this value will not be included in the reconstruction.
thresh_refine 50.0 ! Mode 4: Score threshold above which search will not be performed.
nbootstrap 1000 ! Number of bootstrap volumes to calculate real-space variance map.
FMAG F ! T or F. Set to T to refine particle magnification. Not recommended in most cases.
FDEF F ! T or F. Set to T to refine defocus per micrograph. Not recommended in most cases.
FASTIG F ! T or F. Set to T to refine astigmatism. Not recommended in most cases.
FPART F ! T or F. Set to T to refine defocus for each particle. Not recommended in most cases.
FFILT T ! T or F. Set to T to apply optimal filter to reconstruction. Recommended in most cases.
FMATCH F ! T or F. Set to T to output matching projections. Only needed for diagnostics.
FBEAUT F ! T or F. Set to T to apply symmetry also in real space. Not needed in most cases.
FBOOST T ! T or F. Set to T to allow potential overfitting during refinement. Not recommended in most cases.
RBfactor 0.0 ! B-factor sharpening (when < 0) applied during refinement. Not recommended in most cases.
beam_tilt_x 0.0 ! Beam tilt in mrad along X-axis.
beam_tilt_y 0.0 ! Beam tilt in mrad along y-axis.
mp_cpus 1 ! Number of CPUs to use for each reconstruction job.
restart_after_crash F ! T or F. Set to T to restart job if it crashes.
delete_scratch T ! Delete intermediate files in scratch directory.
qsub_string_ref "" ! String to add to cluster jobs submitted for refinement (only for SGE, SLURM and PBS clusters).
qsub_string_rec "" ! String to add to cluster jobs submitted for reconstruction (only for SGE, SLURM and PBS clusters).
first_particle
last_particle
frealign_bin_dir
scratch_dir
# Masking parameters (for expert users)
mask_file
mask_edge 5 ! Width of cosine edge in pixels to add around mask. Set to 0 to leave mask unchanged.
mask_outside_weight 0.0 ! Factor to downweight density outside of mask (normally 0.0 - 1.0).
mask_filt_res 0.0 ! Filter radius (in A) to low-pass filter density outside density. Set to 0.0 to skip filtering.
mask_filt_edge 5 ! Width of cosine edge in reciprocal pixels to add to filter function.
frealign_calc_reconstructions
hi qwertgfdsa,
If you do a simple frealign_calc_reconstructions, do you get a featureless ball?
This would indicate that something went wrong during conversion, probably during generation of the mrc stack.
How do you do this step?
Best,
Axel
wrong outer_radius
In reply to frealign_calc_reconstructions by Axel
Hi Axel,
realign_calc_reconstructions of cycle 1 give me a normal map.
I figured it out why.
Just a stupid mistake.
My particle has a diameter of 180A
but I input 180 as the outer_radius....
Thank you very much for the help!